Muna Research Articles

FT-IR, 1H, 13C NMR, ESI–MS and semiempirical investigation of the structures of Monensin phenyl urethane complexes with the sodium cation

In this paper three forms of phenyl urethane of Monensin i.e. its acid form (H–MU) and its 1:1 complex with NaClO4 (H–MU–Na) and its sodium salt (Na–MU) were obtained and their structures were studied by FT-IR, 1H and 13C NMR, ESI MS and PM5 methods. The FT-IR data of Na–MU complexes demonstrate that the CO urethane group is not engaged in the complexation of the sodium cation. However spectroscopic studies of H–MU–Na complex show that the structure in which this CO urethane groups participate in the complexation is also present, but it is in the minority. The PM5 semiempirical calculations allow visualisation of all structures and determination of the hydrogen bond parameters.

Bacterial energetics at high pH: what happens to the H+ cycle when the extracellular H+ concentration decreases?

A decrease in the extracellular H+ concentration creates difficulties for membrane-linked energetics in bacteria employing H+ as the coupling ion. At high extracellular pH (pHo), H+ ions pumped from the cell by, say, the respiratory chain, are immediately neutralized by the alkaline extracellular medium. Under such conditions, the only driving force that might compel outer H+ ions to return to cytosol and perform their function is the electric potential difference across the cytoplasmic membrane (delta psi). However, when delta pH in the opposite direction is equal to, e.g., 2 pH units (intracellular pH = 7.5 at pHo = 9.5), delta psi would be so high that the risk of membrane electric breakdown would increase. This is why some bacteria deal with high pH by, for example, replacing H+ by Na+ as the coupling ion rather than by increasing delta psi. It has been shown in several species of bacteria that the alkalinization of the growth medium induces primary Na+ pumps (e.g. Na(+)-motive respiratory chain enzymes and Na+ ATPase). Electrogenic Na+ efflux via these pumps produces an electrochemical Na+ potential difference (delta mu Na+) composed of delta psi and delta pNa+. delta mu Na+ can be used to perform various types of membrane-linked work. The delta psi constituent of delta mu Na+ may maintain electrophoretic influx of H+ such that the alkalinization of cytoplasm is prevented. The latter function may be supported by a mechanism based on the uphill influx of Cl- instead of Na+. This seems to be the case for alkaliphilic and halophilic Natronobacter pharaonis. There is an indication that not only Na+ but also Ca2+ may substitute for H+ in Gleobacter violaceus growing at high pH.

Study of cytochrome bo function in Vitreoscilla using a cyo(-) knockout mutant.

The bacterium, Vitreoscilla, produces a delta mu(Na+) across its membrane during respiration. A key enzyme for this function is the cytochrome bo terminal oxidase which, when incorporated into synthetic proteoliposomes, pumps Na(+) across the membrane upon the addition of a substrate. A Vitreoscilla cytochrome bo knock out (cyo(-)) mutant was isolated by transposon mutagenesis using pUT-mini-Tn5Cm. The membranes of this mutant lacked the characteristic 416 nm peak and 432 nm trough in CO difference spectra, which are clearly visible in spectra of the Vitreoscilla wild-type, but peaks at 627, 560, and 530 nm in reduced minus oxidized difference spectra indicate that cytochrome bd is still present. The specific NADH oxidase and ubiquinol-1 oxidase activities of the cyo(-) mutant membranes were less than those of Vitreoscilla wild-type and Escherichia coli membranes, and the stimulation of these activities of the mutant and E. coli membranes by 75 mM NaCl was approximately 50% less than that of Vitreoscilla wild-type membranes. The ubiquinol-1 oxidase activity of the cyo(-) mutant membranes was inhibited by 10 mM KCN to a lesser degree than that of the Vitreoscilla wild-type and E. coli membranes (50, 80, and 85%, respectively). This result is also consistent with the cyo(-) mutant membrane fragments containing only the cytochrome bd terminal oxidase, which is known to be less sensitive to KCN. Although the maximum respiration and growth of the cyo(-) mutant were less than those of the wild-type, this mutant is still capable of growing with cytochrome bd alone.

Sequence of a Gene Cluster from Malonomonas Rubra Encoding Components of the Malonate Decarboxylase Na+ Pump and Evidence for Their Function

Malonate decarboxylation in Malonomonas rubra involves the formation of malonyl-S-[acyl-carrier protein] from acetyl-S-[acyl-carrier protein] and malonate, carboxyltransfer to a biotin protein and its decarboxylation that is coupled to delta mu Na+ generation. The genes encoding components of the malonate decarboxylase enzyme system have been cloned and sequenced. These are located within a gene cluster of approximately 11 kb comprising 14 genes that have been termed madYZGBAECDHKFLMN in the given order. Upstream of madY an open reading frame pointing into the opposite direction of the mad genes was found with structural similarities to insertion-sequence elements. The upstream region also contains DNA regions which are typical for an Escherichia coli sigma 70 promoter. Within 950 bp downstream of madN no other open reading frame was found. This region contains a putative terminator sequence. The intergenic regions within the mad gene cluster are short (usually < 70 bp, maximum 302 bp) and ribosome binding sites were defined before all 14 genes. Thus, this DNA region could form a transcriptional unit and all 14 genes could be translated into proteins. The genes madABCDEF encode the structural proteins of the malonate decarboxylase as yet identified. By comparing protein and DNA sequences and by data bank searches for related proteins with known function the following assignments could be made: MadA represents the acyl-carrier-protein-transferase component. MadB is the integral membrane-bound carboxybiotin protein decarboxylase, MadC and MadD are the two subunits of the carboxyltransferase, MadE is the acyl carrier protein and MadF is the biotin protein. Sequence comparison further indicates that MadH could be involved in the acetylation of the phosphoribosyl-dephospho-CoA prosthetic group and MadG could be involved in its biosynthesis. MadL and MadM are membrane proteins that could function as malonate carrier. The function of the madY,Z,K and N gene products is as yet unknown.

Contribution of Na+/Ca2+ exchanger in maintaining [Ca2+]c at a stable state in rat pancreatic islets.

The effects of lowering extracellular Na+ concentration [Na+]o, on cytosolic Ca2+ concentration, [Ca2+]c were examined by a microfluorimetric method using fura-2 in perifused preparations of isolated rat pancreatic islets. The total replacement of extracellular Na+ (Na+o) by equimolar N-methyl-D-(--)-glucamine caused a rapid rise in [Ca2+]c, and partial replacement of Na+o resulted in correlative rises in [Ca2+]c in accordance with the magnitude of reduced [Na+]o. The rise in [Ca2+]c induced by Na+o removal was strongly inhibited in the Ca2+o-deficient environment or by Ni2+. The [Ca2+]c rise, however, remained almost unchanged in the presence of nifedipine or SK&F 96365, and was enhanced by the addition of ouabain. The electrochemical gradients for Ca2+ (delta mu Ca2+) and Na+ (delta mu Na+) were calculated to be 39.08 and 12.8 kJ/mol, respectively, in this study, indicating a stoichiometry of 3Na+: 1 Ca2+. These results indicate that, in rat pancreatic islets, the rise in [Ca2+]c induced by lowering [Na+]o is mainly due to Ca2+ entry medicated by the Na+/Ca2+ exchanger operating with the stoichiometry of 3Na+:1 Ca2+, and that the Na+/Ca2+ exchanger plays an important role in maintaining stable-state [Ca2+]c.

Induction of the Escherichia coli cytochrome d by low delta mu H+ and by sodium ions.

Regulation of synthesis of cytochrome d in Escherichia coli has been studied using mutants with cytochrome-d--beta-galactosidase gene fusions. It was shown that various protonophorous uncouplers, when added to the growth medium, cause induction of the cytochrome d synthesis. The cytochrome-d-inducing activity of uncouplers correlates with their ability to inhibit such a delta mu (H+)-driven function as motility of the E. coli cells. An increase in the Na+ concentration in the growth medium from 1.5 mM to 25 mM results in induction of the cytochrome d synthesis. The cytochrome-d-inducing effect of uncouplers is much more pronounced when the Na+ concentration is high than when it is low. These data are in agreement with the assumption that cytochrome d is involved in the Na+ energetics substituting for the H+ energetics when the latter appears to be inefficient. Mutations in arcA or arcB genes (but not in fnr gene) completely prevent the increase in the cytochrome d level induced by uncouplers but are without effect on that induced by Na+. It is assumed that in the control of the cytochrome d synthesis, the Arc system is involved in the delta mu H+ sensing whereas sensing of delta mu Na+ (or of the Na+ concentration) is mediated by some other receptor system.

Delta mu Na+ drives the synthesis of ATP via an delta mu Na(+)-translocating F1F0-ATP synthase in membrane vesicles of the archaeon Methanosarcina mazei Gö1.

Methanosarcina mazei Gö1 couples the methyl transfer from methyl-tetrahydromethanopterin to 2-mercaptoethanesulfonate (coenzyme M) with the generation of an electrochemical sodium ion gradient (delta mu Na+) and the reduction of the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreoninephosphate with the generation of an electrochemical proton gradient (delta muH+). Experiments with washed inverted vesicles were performed to investigate whether both ion gradients are used directly for the synthesis of ATP. delta mu Na+ and delta mu H+ were both able to drive the synthesis of ATP in the vesicular system. ATP synthesis driven by heterodisulfide reduction (delta mu H+) or an artificial delta pH was inhibited by the protonophore SF6847 but not by the sodium ionophore ETH157, whereas ETH157 but not SF6847 inhibited ATP synthesis driven by a chemical sodium ion gradient (delta pNa) as well as the methyl transfer reaction (delta mu Na+). Inhibition of the Na+/H+ antiporter led to a stimulation of ATP synthesis driven by the methyl transfer reaction (delta mu Na+), as well as by delta pNa. These experiments indicate that delta mu Na+ and delta mu H+ drive the synthesis of ATP via an Na(+)- and an H(+)-translocating ATP synthase, respectively. Inhibitor studies were performed to elucidate the nature of the ATP synthase(s) involved. delta pH-driven ATP synthesis was specifically inhibited by bafilomycin A1, whereas delta pNa-driven ATP synthesis was exclusively inhibited by 7-chloro-4-nitro-2-oxa-1,3-diazole, azide, and venturicidin. These results are evidence for the presence of an F(1)F(0)-ATP synthase in addition to the A(1)A(0)-ATP synthase in membranes of M. Mazei Gö1 and suggest that the F(1)F(0)-type enzyme is an Na+-translocating ATP synthase, whereas the A(1)A(0)-ATP synthase uses H+ as the coupling ion.

Bacterial sodium ion-coupled energetics.

For many bacteria Na+ bioenergetics is important as a link between exergonic and endergonic reactions in the membrane. This article focusses on two primary Na+ pumps in bacteria, the Na(+)-translocating oxaloacetate decarboxylase of Klebsiella pneumoniae and the Na(+)-translocating F1Fo ATPase of Propionigenium modestum. Oxaloacetate decarboxylase is an essential enzyme of the citrate fermentation pathway and has the additional function to conserve the free energy of decarboxylation by conversion into a Na+ gradient. Oxaloacetate decarboxylase is composed of three different subunits and the related methylmalonyl-CoA decarboxylase consists of five different subunits. The genes encoding these enzymes have been cloned and sequenced. Remarkable are large areas of complete sequence identity in the integral membrane-bound beta-subunits including two conserved aspartates that may be important for Na+ translocation. The coupling ratio of the decarboxylase Na+ pumps depended on delta muNa+ and decreased from two to zero Na+ uptake per decarboxylation event as delta mu Na+ increased from zero to the steady state level. In P. modestum, delta mu Na+ is generated in the course of succinate fermentation to propionate and CO2. This delta mu Na+ is used by a unique Na(+)-translocating F1Fo ATPase for ATP synthesis. The enzyme is related to H(+)-translocating F1Fo ATPases. The Fo part is entirely responsible for the coupling of ion specificity. A hybrid ATPase formed by in vivo complementation of an Escherichia coli deletion mutant was completely functional as a Na(+)-ATP synthase conferring the E. coli strain the ability of Na(+)-dependent growth on succinate. The hybrid consisted of subunits a, c, b, delta and part of alpha from P. modestum and of the remaining subunits from E. coli. Studies on Na+ translocation through the Fo part of the P. modestum ATPase revealed typical transporter-like properties. Sodium ions specifically protected the ATPase from the modification of glutamate-65 in subunit c by dicyclohexylcarbodiimide in a pH-dependent manner indicating that the Na+ binding site is at this highly conserved acidic amino acid residue of subunit c within the middle of the membrane.

On the mechanism of sodium ion translocation by oxaloacetate decarboxylase of Klebsiella pneumoniae.

Proteoliposomes reconstituted with purified oxaloacetate decarboxylase of Klebsiella pneumoniae catalyzed the uptake of Na+ ions upon oxaloacetate decarboxylation. The degree of coupling between the chemical and the vectorial reaction is dependent on the reconstitution conditions, and with the best preparations approaches a stoichiometry of two Na+ ions per decarboxylation of one oxaloacetate. This coupling ratio is observed only in the absence of a delta mu Na+, immediately after oxaloacetate addition. The ratio gradually declines during development of the electrochemical Na+ ion gradient and becomes zero in the steady state. The Na+ pump, however, continued to decarboxylate oxaloacetate and to catalyze Na+ influx at the apparent stoichiometry of two Na+ ions per decarboxylation event. During the steady state, this influx must be compensated by Na+ efflux of the same size. The efflux is catalyzed by the Na+ pump upon oxaloacetate decarboxylation, because in the absence of the substrate the efflux rate dropped to less than 10%. Proteoliposomes loaded with Na2SO4 catalyzed a bicarbonate-dependent uptake of 22Na+ that was completely abolished after incubation with avidin. These results suggest coupling of Na+ translocation to the carboxylation/decarboxylation of the biotin prosthetic group without the requirement for the oxaloacetate/pyruvate interconversion. The oxaloacetate-dependent transport of Na+ into proteoliposomes was inhibited by the additional presence of the beta + gamma subunits of oxaloacetate decarboxylase. A model of Na+ translocation by oxaloacetate decarboxylase based on these experimental results is proposed.

Pollutant-induced depression of the transmembrane sodium gradient in muscles of mussels.

This study deals with the effects of chemical pollutants on the transmembrane potential difference for sodium (delta mu Na) in smooth muscle cells of Mytilus edulis. A method for indirect determination of extracellular space, intracellular ion concentrations and delta mu Na has been developed and is applied in the investigations. The determination is based on concentration data from haemolymph and muscle tissue samples. The precision of the method used was tested by direct measurements of the apparent intracellular concentration of sodium and the membrane potential. On the basis of these tests, the method was evaluated as reasonably good. The method was used to study the sensitivity of the transmembrane delta mu Na in Mytilus edulis to 96 h exposures to various sublethal concentrations of formaldehyde, methanol and mercury. Both formaldehyde and mercury induced a depression of delta mu Na. The observed depressions could be ascribed to a change in both the electrogenic and the chemical components of delta mu Na. A depression of delta mu Na was associated with subsequent clinical injury and death. Methanol did not cause death or any changes in delta mu Na. Because of the observed correlation between depression of delta mu Na and clinical injury, delta mu Na is suggested to have a potential as an indicator of toxicity.

NADH formation by Na(+)-coupled reversed electron transfer in Klebsiella pneumoniae.

Citrate is fermented by Klebsiella pneumoniae to 2 acetate, 0.5 formate and 1.2 CO2. The formation of less than 1 formate and greater than 1 CO2 per citrate can be accounted for by the oxidation of formate to CO2 in order to provide reducing equivalents for the assimilation of citrate into cell carbon. A membrane-bound electron transport chain is apparently involved in NADH synthesis by these cells. The electrons from formate oxidation to CO2 are used to reduce ubiquinone to ubiquinol by membrane-bound formate dehydrogenase and ubiquinol further delivers its electrons to NAD+, if this endergonic reaction is powered by delta mu Na+. The endogenous NADH level of K. pneumoniae cells thus increased in the presence of formate in response to a delta pNa+ greater than -100 mV. NADH formation was completely abolished in the presence of oxygen or after addition of hydroxyquinoline-N-oxide, a specific inhibitor of the Na(+)-translocating NADH:ubiquinone oxidoreductase. The increase of endogenous NADH was dependent on the delta pNa+ applied to the cells. Inverted membrane vesicles of K. pneumoniae catalysed the reduction of NAD+ to NADH with formate as electron donor after application of delta mu Na+ of about 120 mV consisting of delta pNa+ of 60 mV and delta psi of the same magnitude. Neither the delta pNa+ nor the delta psi of this size alone was sufficient to drive the endergonic reaction. Strictly anaerobic conditions were required for NADH formation and hydroxyquinoline-N-oxide completely inactivated the reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

On the mechanism of sodium ion translocation by methylmalonyl‐CoA decarboxylase from Veillonella alcalescens

Veillonella alcalescens during lactate degradation developed an Na+ concentration gradient with 7-8 times higher external than internal Na+ concentrations in the logarithmic growth phase. The gradient declined to a factor of 1.9 in the late stationary phase. Methylmalonyl-CoA decarboxylase reconstituted into proteoliposomes performed an active electrogenic Na+ transport, creating delta psi of 60 mV, delta pNa+ of 50 mV, and delta mu Na+ of 110 mV. In the initial phase of the transport, the decarboxylase catalyzed the uptake of 2 Na+ ions malonyl-CoA molecule decarboxylated. During further development of the electrochemical Na+ gradient, this ratio gradually declined to zero, when decarboxylation continued without further increase of the internal Na+ concentration. The rate of malonyl-CoA decarboxylation declined initially during development of the membrane potential, but remained unchanged later on. Monensin abolished the Na+ gradient and increased the malonyl-CoA decarboxylation rate 2.8-fold. On dissipating the membrane potential with valinomycin, the internal Na+ concentration reached three times higher values than in its absence, and the decarboxylation rate increased 2.8-fold. Methylmalonyl-CoA decarboxylase catalyzed an exchange of internal and external Na+ ions in addition to net Na+ accumulation. The initial rate of Na+ influx was double that of malonyl-CoA decarboxylation. In the following, both rates decreased about twofold in parallel to values which remained constant during further development of the electrochemical Na+ gradient. Thus, Na+ influx and malonyl-CoA decarboxylation follow a stoichiometry of approximately 2:1, independent of the magnitude of the electrochemical Na+ gradient and are thus highly coupled events.

Sodium ion-dependent amino acid transport in membrane vesicles of Bacillus stearothermophilus.

Amino acid transport in membrane vesicles of Bacillus stearothermophilus was studied. A relatively high concentration of sodium ions is needed for uptake of L-alanine (Kt = 1.0 mM) and L-leucine (Kt = 0.4 mM). In contrast, the Na(+)-H(+)-L-glutamate transport system has a high affinity for sodium ions (Kt less than 5.5 microM). Lithium ions, but no other cations tested, can replace sodium ions in neutral amino acid transport. The stimulatory effect of monensin on the steady-state accumulation level of these amino acids and the absence of transport in the presence of nonactin indicate that these amino acids are translocated by a Na+ symport mechanism. This is confirmed by the observation that an artificial delta psi and delta mu Na+/F but not a delta pH can act as a driving force for uptake. The transport system for L-alanine is rather specific. L-Serine, but not L-glycine or other amino acids tested, was found to be a competitive inhibitor of L-alanine uptake. On the other hand, the transport carrier for L-leucine also translocates the amino acids L-isoleucine and L-valine. The initial rates of L-glutamate and L-alanine uptake are strongly dependent on the medium pH. The uptake rates of both amino acids are highest at low external pH (5.5 to 6.0) and decline with increasing pH. The pH allosterically affects the L-glutamate and L-alanine transport systems. The maximal rate of L-glutamate uptake (Vmax) is independent of the external pH between pH 5.5 and 8.5, whereas the affinity constant (Kt) increases with increasing pH. A specific transport system for the basic amino acids L-lysine and L-arginine in the membrane vesicles has also been observed. Transport of these amino acids occurs most likely by a uniport mechanism.

Effects of low extracellular Ca2+ on cytosolic free Ca2+, Na+, and pH of MDCK cells

The effects of lowering the Ca2+ electrochemical potential (delta mu Ca2+) by removing extracellular calcium (Ca2+o) was studied in Madin-Darby canine kidney (MDCK) cells. We measured cytosolic free calcium (Ca2+i), intracellular Na+ (Na+i), and intracellular pH (pHi) as well as Ca2+ and Na+ influx and efflux and H+ secretion. As soon as Ca2+o was lowered to zero, Ca2+ efflux increased 2-fold, and Na+ influx increased 4.3-fold. As a result, Ca2+i fell 71% from 120 to 34.7 nM, and Na+i rose 56% from 16.7 to 26.1 mM. At the same time, H+ secretion was depressed 38%, and pHi fell from 7.32 to 7.22. When Ca2+o was restored to normal (1.3 mM), there was a sharp rise in Ca2+i from 34.7 to 243 nM (+600%) caused by an immediate increase in Ca2+ influx (+112%). At the same time, Na+ efflux increased 20%. Ten to fifteen minutes after restoring Ca2+o to 1.3 mM, Ca2+i, Na+i, and pHi were back to control levels. These results suggest that reducing delta mu Ca2+ activates the forward mode of Na(+)-Ca2+ exchange which leads to a fall in Ca2+i and a decreased delta mu Na+ by increasing Na+i. The fall in delta mu Na+ secondarily depresses Na(+)-H+ exchange which results in a reduced H+ secretion and a fall in pHi. When Ca2+o is restored to 1.3 mM, the reverse mode of Na(+)-Ca2+ exchange is activated, since delta mu Ca2+ is larger than normal because of the low Ca2+i, and delta mu Na+ is reduced because of the high Na+i.(ABSTRACT TRUNCATED AT 250 WORDS)

The sodium cycle in methanogenesis

CH4 formation from CO2 and H2 rather than from formaldehyde and H2 in methanogenic bacteria is inhibited by uncouplers, indicating that CO2 reduction to the formaldehyde level is energy-driven. We report here that in Methanosarcina barkeri the driving force is a primary electrochemical sodium potential (delta mu Na+) generated by formaldehyde reduction to CH4. This is concluded from the following findings. 1. CO2 reduction to CH4 was insensitive towards protonophores, when the Na+/H+ antiporter was inhibited; under these conditions delta mu Na+ was 120 mV (inside negative), whereas both delta mu H+ and the cellular ATP content were low. 2. CO2 reduction to CH4, rather than formaldehyde reduction, was sensitive towards Na+ ionophores, which dissipated delta mu Na+. 3. CO2 reduction to CH4, in the presence of protonophores and Na+/H+ antiport inhibitors, was coupled with the extrusion of 1-2 mol Na+/mol CH4, and formaldehyde reduction to CH4 was coupled with the extrusion of 3-4 mol Na+/mol CH4. Thus during CO2 reduction to the formaldehyde level 2-3 mol Na+ were consumed.

The sodium cycle: a novel type of bacterial energetics.

The progress of bioenergetic studies on the role of Na+ in bacteria is reviewed. Experiments performed over the past decade on several bacterial species of quite different taxonomic positions show that Na+ can, under certain conditions, substitute for H+ as the coupling ion. Various primary Na+ pumps (delta mu Na+ generators) are described, i.e., Na+ -motive decarboxylases, NADH-quinone reductase, terminal oxidase, and ATPase. The delta mu Na+ formed is shown to be consumed by Na+ driven ATP-synthase, Na+ flagellar motor, numerous Na+, solute symporters, and the methanogenesis-linked reverse electron transfer system. In Vibrio alginolyticus, it was found that delta mu Na+, generated by NADH-quinone reductase, can be utilized to support all three types of membrane-linked work, i.e., chemical (ATP synthesis), osmotic (Na+, solute symports), and mechanical (rotation of the flagellum). In Propionigenum modestum, circulation of Na+ proved to be the only mechanism of energy coupling. In other species studied, the Na+ cycle seems to coexist with the H+ cycle. For instance, in V. alginolyticus the initial and terminal steps of the respiratory chain are Na+ - and H+ -motive, respectively, whereas ATP hydrolysis is competent in the uphill transfer of Na+ as well as of H+. In the alkalo- and halotolerant Bacillus FTU, there are H+ - and Na+ -motive terminal oxidases. Sometimes, the Na+ -translocating enzyme strongly differs from its H+ -translocating homolog. So, the Na+ -motive and H+ -motive NADH-quinone reductases are composed of different subunits and prosthetic groups. The H+ -motive and Na+ -motive terminal oxidases differ in that the former is of aa3-type and sensitive to micromolar cyanide whereas the latter is of another type and sensitive to millimolar cyanide. At the same time, both Na+ and H+ can be translocated by one and the same P. modestum ATPase which is of the F0F1-type and sensitive to DCCD. The sodium cycle, i.e., a system composed of primary delta mu Na+ generator(s) and delta mu Na+ consumer(s), is already described in many species of marine aerobic and anaerobic eubacteria and archaebacteria belonging to the following genera: Vibrio, Bacillus, Alcaligenes, Alteromonas, Salmonella, Klebsiella, Propionigenum, Clostridium, Veilonella, Acidaminococcus, Streptococcus, Peptococcus, Exiguobacterium, Fusobacterium, Methanobacterium, Methanococcus, Methanosarcina, etc. Thus, the "sodium world" seems to occupy a rather extensive area in the biosphere.

Na+ electrochemical gradient and Na+-Ca2+ exchange in rat proximal tubule.

The basolateral cell membrane of the rat proximal tubule contains a Na+-Ca2+ exchanger that may participate in the regulation of cytosolic calcium (Cai) and Ca2+ transport. In this work, the activity and orientation of the Na+-Ca2+ exchanger was studied in rat proximal tubules. The experiments were based on the thermodynamic notion that the exchanger is driven by the prevalence of either of two electrochemical gradients, that for Na+ (delta mu Na+) or for Ca2+ (delta mu Ca2+). Reductions in delta mu Na+, achieved by lowering extracellular Na+ (Nao) from 150 to 15 mM, increased Cai, decreased 45Ca efflux, and increased 45Ca influx. These changes occurred concurrently. When delta mu Na+ was reduced by increasing intracellular Na+ (Nai) with 10(-3) M oubain, Cai also increased. The effect of ouabain was probably dependent on Nai accumulation because the surge in Cai was prevented by exposure of the tubules to 5 mM Nao before ouabain exposure. On the other hand, when delta mu Na+ was lowered mM Nao and then by reducing Nao to 15 mM, Cai rose in two additive stages. We conclude from these data that in the rat proximal tubule the basal state of the Na+-Ca2+ exchanger is in forward mode, Nao-Cai. Moreover, the function of the Na+-Ca2+ exchanger is in accord with predictions derived from a thermodynamic analysis of its function.

ATP synthesis is driven by an imposed delta pH or delta mu H+ but not by an imposed delta pNa+ or delta mu Na+ in alkalophilic Bacillus firmus OF4 at high pH.

Starved whole cells of alkalophilic Bacillus firmus OF4 that are equilibrated at either pH 10.2, 9.5, or 8.5 synthesize ATP in response to a pH gradient that is imposed by rapid dilution of the cyanide-treated cells into buffer at pH 7.5. If a valinomycin-mediated potassium diffusion potential (positive out) is generated simultaneously with the pH gradient, then the rate of ATP synthesis and the level of synthesis achieved is much higher than upon imposition of a pH gradient alone. By contrast, imposition of a large chemical gradient of Na+, either in the presence or absence of a concomitant diffusion potential, fails to result in ATP synthesis. We conclude that this organism does not possess a sodium-motive ATPase that can be made to synthesize detectable levels of ATP by imposition of a suitably large chemical or electrochemical gradient of Na+. On the other hand, a proton-translocating ATPase is in evidence when protons are provided at very high pH, corroborating our earlier work on extremely alkalophilic bacilli. Oxidative phosphorylation must, then, be catalyzed in these organisms by a proton-translocating ATPase even though the putative bulk driving forces for such a catalyst are low under optimal growth conditions. Stable, imposed pH gradients of 1 unit, comparable to the magnitude of the total electrochemical proton gradient of growing cells, result in much lower ATP concentrations than observed in such cells. We hypothesize that ATP synthesis in growing cells utilizes protons that are made available by some localized pathway between proton pumps and the ATP synthase.

(Na + K + 2Cl) cotransport in cultured embryonic chick heart cells.

The coupled movements of Na, K, and Cl were studied in cultured chick embryonic heart cells using ion-selective microelectrodes. Movements of K and Cl in response to changes in extracellular [K] ([K]o) showed a furosemide-sensitive coupled process. The movement of Na was then studied. Lowering extracellular [Na] ([Na]o) to 27 mM caused a decrease in intracellular Cl activity (aicl). Upon restoring [Na]o to 143 mM, Cl was taken up against its electrochemical gradient (delta mu Cl). In Cl-free solution, cells lost Na against delta mu Na and simultaneously lost Cl. Upon restoring extracellular [Cl] ([Cl]o), Cl was taken up against delta mu Cl; this was accompanied by an uptake of Na. The Cl uptake was 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS)-insensitive (0.1 mM) but inhibited by removing Nao. Both Cl and Na uptakes were potentiated by raising [K]o from 5.4 to 15 mM, and Na uptake was diminished by lowering [K]o to 1 mM. In all experiments, Cl and Na movements were furosemide (0.3 mM) or bumetanide-sensitive (0.1 mM). Removal of Nao, with resultant depletion of intracellular [Na] ([Na]i), blocked the furosemide or bumetanide-sensitive Cl loss or uptake upon exposure to zero or 133 mM [K]o + SITS (0.1 mM), respectively. These results suggest that cultured heart cells possess an electroneutral (Na + K + 2Cl) cotransport.

Sodium‐selective micro‐electrode study of apical permeability in frog skin: effects of sodium, amiloride and ouabain.

The intracellular sodium ion activity (aiNa), apical membrane potential (psi ac) and apical sodium electrochemical driving force (delta mu Na) in Rana temporaria skin were measured using double-barrelled sodium-sensitive micro-electrodes, in the presence of various apical sodium activities (aoNa), amiloride, ouabain, and during voltage clamp of psi ac. The permeability and specific conductance of the apical cell membrane to sodium entry (PaNa and GaNa respectively) were calculated from the Goldman-Hodgkin-Katz equation and the Nernst-Planck (electrodiffusion) permeability equations respectively. The roles of aoNa and aiNa in the control of apical sodium entry were studied. PaNa increased linearly with log decrease in aoNa between 79 and 0.01 mM. Under short-circuit conditions, aiNa remained constant over the aoNa range of 10-79 mM, but decreased when aoNa was lower than 10 mM, due to a fall in delta mu Na and GaNa. Amiloride decreased PaNa, GaNa and aiNa, a result analogous to that observed in spontaneous low-transporting skins. Ouabain inhibited sodium transport and increased aiNa before any changes in PaNa occurred. The latter decreased only when aiNa rose above 15 mM. Increasing delta mu Na by hyperpolarizing voltage clamp of the apical cell membrane elicited a saturable increase in aiNa. The opposite effect was elicited by depolarizing psi ac. Electrodiffusion appears to be the sole mode of apical sodium entry.